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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had a tremendous impact worldwide. Mapping virus-host interactions is critical to understand disease progression. MicroRNAs (miRNAs) are important RNA regulators, but their interaction with SARS-CoV-2 RNA was not experimentally investigated. Here, using Argonaute (AGO) cross-linking immunoprecipitation combined with RNA proximity ligation (CLEAR-CLIP), we provide unbiased mapping of SARS-CoV-2/miRNA interactions. We identified six main regions on the viral RNA bound primarily by one specific miRNA. Targeted mutagenesis and AGO1-3 knockdown demonstrated that these interactions are not critical for virus production. Moreover, we identified perturbed regulation of cellular miRNA interactions during infection, including non-compensated viral sequestration of the miR-15 family. Transcriptome analysis further showed that mRNAs targeted by this miRNA family are derepressed. This work delineates the interphase between miRNA regulation and SARS-CoV-2 infection and further contributes to deciphering the full molecular interactome of this virus.

Ribavirin remains relevant for successful treatment of chronic hepatitis C virus (HCV) infections in low-income settings, as well as for therapy of difficult-to-treat HCV patients. We studied the effect of ribavirin against cell-culture adapted HCV of genotypes 1, 2 and 3, representing ~80% of global infections. TNcc(1a) was the most sensitive to ribavirin, while J6/JFH1(2a) was the most resistant. EC50s ranged from 21 μM (95%CI: 20–22 μM) to 189 μM (95%CI: 173–207 μM). Substitutions at position 415 of NS5B resulted in little or no change to ribavirin sensitivity (0.7–0.9 fold) but conferred moderate drug resistance during extended treatment of genotype 1 (1.8-fold). NS5A and NS5B sequences could alter ribavirin sensitivity 2-4-fold, although their contribution was not simply additive. Finally, we detected limited accumulation of mutations associated with ribavirin treatment. Our findings show that the antiviral effect of ribavirin on HCV is strain-dependent and is influenced by the specific sequence of multiple HCV nonstructural proteins.

Background & Aims Protease inhibitors (PIs) are of central importance in the treatment of patients with chronic hepatitis C virus (HCV) infection. HCV NS3 protease (NS3P) position 80 displays polymorphisms associated with resistance to the PI simeprevir for HCV genotype 1a. We investigated the effects of position-80-substitutions on fitness and PI-resistance for HCV genotypes 1-6, and analyzed evolutionary mechanisms underlying viral escape mediated by pre-existing Q80K. Methods The fitness of infectious NS3P recombinants of HCV genotypes 1-6, with engineered position-80-substitutions, was studied by comparison of viral spread kinetics in Huh-7.5 cells in culture. Median effective concentration (EC50) and fold resistance for PIs simeprevir, asunaprevir, paritaprevir, grazoprevir, glecaprevir and voxilaprevir were determined in short-term treatment assays. Viral escape was studied by long-term treatment of genotype 1a recombinants with simeprevir, grazoprevir, glecaprevir and voxilaprevir and of genotype 3a recombinants with glecaprevir and voxilaprevir, next generation sequencing, NS3P substitution linkage and haplotype analysis. Results Among tested PIs, only glecaprevir and voxilaprevir showed pan-genotypic activity against the original genotype 1-6 culture viruses. Variants with position-80-substitutions were all viable, but fitness depended on the specific substitution and the HCV isolate. Q80K conferred resistance to simeprevir across genotypes but had only minor effects on the activity of the remaining PIs. For genotype 1a, pre-existing Q80K mediated accelerated escape from simeprevir, grazoprevir and to a lesser extent glecaprevir, but not voxilaprevir. For genotype 3a, Q80K mediated accelerated escape from glecaprevir and voxilaprevir. Escape was mediated by rapid and genotype-, PI- and PI-concentration-dependent co-selection of clinically relevant resistance associated substitutions. Conclusions Position-80-substitutions had relatively low fitness cost and the potential to promote HCV escape from clinically relevant PIs in vitro, despite having a minor impact on results in classical short-term resistance assays. Lay summary Among all clinically relevant hepatitis C virus protease inhibitors, voxilaprevir and glecaprevir showed the highest and most uniform activity against cell culture infectious hepatitis C virus with genotype 1-6 proteases. Naturally occurring amino acid changes at protease position 80 had low fitness cost and influenced sensitivity to simeprevir, but not to other protease inhibitors in short-term treatment assays. Nevertheless, the pre-existing change Q80K had the potential to promote viral escape from protease inhibitors during long-term treatment by rapid co-selection of additional resistance changes, detected by next generation sequencing.

The six major epidemiologically important hepatitis C virus (HCV) genotypes differ in global distribution and antiviral responses. Full-length infectious cell-culture adapted clones, the gold standard for HCV studies in vitro, are missing for genotypes 4 and 5. To address this challenge for genotype 5, we constructed a consensus full-length clone of strain SA13 (SA13fl), which was found non-viable in Huh7.5 cells. Step-wise adaptation of SA13fl-based recombinants, beginning with a virus encoding the NS5B-thumb domain and 3´UTR of JFH1 (SA13/JF372-X), resulted in a high-titer SA13 virus with only 41 JFH1-encoded NS5B-thumb residues (SA13/JF470-510cc); this required sixteen cell-culture adaptive substitutions within the SA13fl polyprotein and two 3´UTR-changes. SA13/JF372-X and SA13/JF470-510cc were equally sensitive to nucleoside polymerase inhibitors, including sofosbuvir, but showed differential sensitivity to inhibitors targeting the NS5B palm or thumb. SA13/JF470-510cc represents a model to elucidate the influence of HCV RNA elements on viral replication and map determinants of sensitivity to polymerase inhibitors.

Background & Aims We investigated antibody responses to hepatitis C virus (HCV) antigens E1 and E2 and the relevance of animal models for vaccine development. We compared antibody responses to vaccination with recombinant E1E2 complex in healthy volunteers, non-human primates (NHPs), and mice. Methods We analyzed 519 serum samples from participants in a phase 1 vaccine trial (ClinicalTrials.gov identifier NCT00500747) and compared them with serum or plasma samples from C57BL/6J mice (n = 28) and rhesus macaques (n = 4) immunized with the same HCV E1E2 antigen. Blood samples were collected at different time points and analyzed for antibody binding, neutralizing activity, and epitope specificity. Monoclonal antibodies from the immunized NHPs were isolated from single plasmablasts and memory B cells, and their immunogenetic properties were characterized. Results Antibody responses of the volunteers, NHPs, and mice to the non-neutralizing epitopes on the E1 N-terminus and E2 hypervariable region 1 did not differ significantly. Antibodies from volunteers and NHPs that neutralized heterologous strains of HCV primarily interacted with epitopes in the antigen region 3. However, the neutralizing antibodies were not produced in sufficient levels for broad neutralization of diverse HCV isolates. Broadly neutralizing antibodies similar to the human VH1-69 class antibody specific for antigen region 3 were produced in the immunized NHPs. Conclusions In an analysis of vaccinated volunteers, NHPs, and mice, we found that recombinant E1E2 vaccine antigen induces high-antibody titers that are insufficient to neutralize diverse HCV isolates. Antibodies from volunteers and NHPs bind to the same neutralizing epitopes for virus neutralization. NHPs can therefore be used as a preclinical model to develop HCV vaccines. These findings also provide useful baseline values for development of vaccines designed to induce production of neutralizing antibodies.

Genetic recombination is an important evolutionary mechanism for RNA viruses. The significance of this phenomenon for hepatitis C virus (HCV) has recently become evident, with the identification of circulating recombinant forms in HCV-infected individuals and by novel data from studies permitted by advances in HCV cell culture systems and genotyping protocols. HCV is readily able to produce viable recombinants, using replicative and non-replicative molecular mechanisms. However, our knowledge of the required molecular mechanisms remains limited. Understanding how HCV recombines might be instrumental for a better monitoring of global epidemiology, to clarify the virus evolution, and evaluate the impact of recombinant forms on the efficacy of oncoming combination drug therapies. For the latter, frequency and location of recombination events could affect the efficacy of multidrug regimens. This review will focus on current data available on HCV recombination, also in relation to more detailed data from other RNA viruses.

Hepatitis C virus (HCV) purification by ultracentrifugation is difficult because of the low and heterogeneous density of native and cultured viruses. It was recently shown that inserting flag tag into envelope protein 2 (E2) of HCV permitted virus purification by affinity chromatography. However, flag-tagged viruses had drastically altered properties, and purification yield was low. In this study, we found that insertion of flag tag at the N-terminus of E2 in HCV recombinant J6/JFH1 did not affect viability in Huh7.5 cells, and that flag-tagged virus had physiochemical properties similar to the original virus. Flag-tagged virus was susceptible to flag-specific antibody neutralization, and infected cells could be immuno-stained by anti-flag antibodies. Using affinity chromatography with anti-flag resin we repeatedly obtained ~30% recovery of infectious particles. The full viability and unaltered physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development.

Background & Aims Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. Methods The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. We examined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments. Results Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equally sensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. Conclusions Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS. These features of HCV genotype 6a could compromise treatment.

Genetic polymorphisms seem to influence the response on antidepressant treatment and moderate the impact of stress on depression. The present study aimed to assess, whether allelic variants and stressful life events interact on the clinical outcome of depression. In a sample of 290 systematically recruited patients diagnosed with a single depressive episode according to ICD-10, we assessed the outcome of antidepressant treatment and the presence of stressful life events in a 6-month period preceding onset of depression by means of structured interviews. Further, we genotyped nine polymorphisms in the genes encoding the serotonin transporter, brain derived neurotrophic factor, catechol-O-methyltransferase, angiotensin converting enzyme, tryptophan hydroxylase, and the serotonin receptors 1A, 2A, and 2C. We found no evidence that the effects of the genetic polymorphisms on treatment outcome were dependent on stressful life events experienced by the individual prior to onset of depression.

The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover, NTRNS2 a chimeric virus equivalent to the previously reported FL-J6/JFH chimera, showed a 10-fold enhancement of virus titers compared to CNS2. NTRNS2 differs from CNS2 by three nucleotide differences residing in the 5′ NTR and core coding sequence and all three nucleotide changes were necessary for increased virion production. Importantly, cells producing Cp7 virus showed increased apoptosis compared with JFH1, an effect correlating with virion production. These studies begin to unravel requirements for robust virus replication and the relationship between increased virion production and host cell viability.

Background & Aims: Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a, which has a high prevalence worldwide. Methods: Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies. Results: S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NS5A. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants with individual mutations (in p7 and NS3, respectively) were fully viable without the requirement for additional mutations. The biological relevance of our system was shown by studying dependence of 3a/JFH1 infection on CD81, and its impact on distribution of intracellular lipids. Conclusions: We developed a robust intergenotypic recombinant cell culture system for HCV genotype 3a, providing a valuable tool for studies of 3a core-NS2 and related therapeutics.

GBV-B, a member of the Flaviviridae family of viruses, is the virus most closely related to HCV, and GBV-B infection in tamarin monkeys might represent a valuable surrogate animal model of HCV infection. In the current study, GBV-B was successfully transmitted to two marmosets (Callithrix jaccus). The infection resulted in viremia of 14- and 17-week duration, respectively, and was accompanied by elevation of isocitrate dehydrogenase activity. These data confirm that marmosets might represent an attractive model for GBV-B infection. The sequence of GBV-B NS5A, which was previously reported to have one of the highest mutation rates during infection in tamarins, was determined for viruses recovered from the inoculum and from marmoset blood samples obtained at weeks 1, 8, and 14 post inoculation in one marmoset and at weeks 2, 8, and 17 post inoculation in the other marmoset. In both animals, we detected four substitutions (R1945K, K2052G, F2196L, and G2268E), in the virus recovered immediately before viral clearance. Interestingly, two of these mutations (F2196L and G2268E) were described recently for viruses recovered from persistently infected tamarins. Appearance of these mutations presumably reflects a mechanism of immune escape rather than adaptation of the virus to a new host.

DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers. These results may have implications for the design and development of HCV DNA vaccines.

Hepatitis C virus (HCV) is a member of the Flaviviridae family.124 The Flaviviridae family of viruses are associated with human and animal diseases and encompass at least three different genera: pestiviruses, such as bovine viral diarrhea virus and classic swine fever virus that cause disease in cattle and pigs, respectively; flaviviruses, the most important cause of arthropod-transmitted viral diseases, for example, dengue fever and yellow fever; and hepacivirus, whose sole member is HCV. The GB viruses, which are also members of the Flaviviridae, are most closely related to HCV and infect humans and monkeys.13,89,144,145 One of the signature characteristics of HCV is its genetic heterogeneity.16 Thus, HCV isolates from around the world have been divided into six major genotypes and more than 100 subtypes.16,45 Moreover, genetic heterogeneity of HCV within infected individuals is manifested as a mixture of closely related but distinct genomes called a quasispecies.16,100 More than 2% of the world population is chronically infected with HCV.66,120,170 In developed countries, HCV is the main cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; HCV-related end-stage liver disease is now the leading reason for liver transplantation.109 Hepatitis C virus infection is also associated with a number of extrahepatic diseases, such as cryoglobulinemia and glomerulonephritis.65 The most important feature of HCV infection is its high rate of chronicity. More than 80% of HCV-infected individuals develop a chronic infection.66 Unfortunately, treatment of chronic hepatitis C with interferon (IFN), the principal recognized therapeutic agent, succeeds only in 20% of the cases.109 The response rate is even lower in patients infected with genotype 1 HCV. Recently, a combination treatment of IFN and ribavirin has achieved higher response rates both in patients infected with genotype 1 and in patients infected with other genotypes.101,118 Despite the heartening progress achieved in treating chronic hepatitis C with combination therapy, the overall response rate in these patients is still below 50%. A better understanding of the molecular biology of HCV has highlighted targets for new therapeutic strategies. The genetic heterogeneity of HCV is a major confounding factor; thus, the design of effective drugs might be facilitated by targeting conserved regions of HCV. Drugs directed against conserved sequences of the 5′ untranslated region (UTR)8,53,123,130 and the serine-protease domain within NS328,84 have already been tested in vitro. The lack of a reliable cell culture system or small animal model for HCV (the chimpanzee is the only recognized animal model) presents significant obstacles for the study of these new therapeutic agents. The recent availability of infectious HCV cDNA clones80,172,174 may facilitate the establishment of useful in vitro replication systems.

GB virus-B (GBV-B) is a member of the Flaviviridae family of viruses. This RNA virus infects tamarins, but its natural host is not known. GBV-B has special interest because it is the virus that is most closely related to hepatitis C virus (HCV), an important human pathogen. In the present study, we identified a previously unrecognized sequence at the 3' end of the GBV-B genome. This new 3' terminal sequence can form several predicted stem-loop structures as is typical for other members of the Flaviviridae family. We constructed molecular clones and showed that the new 3' UTR sequence was critical for in vivo infectivity. After intrahepatic transfection of two tamarins with RNA transcripts of the full-length GBV-B clone, we detected high viral titers from Week 1 postinoculation with peak titers of approximately 10(8) genome equivalents/ml. The viremic pattern of GBV-B infection in the transfected animals was the same as in animals inoculated intravenously with the virus pool used as the cloning source. The sequence of the recombinant virus was recovered from one of the tamarins and shown to be identical to that of the infectious clone. The development of severe hepatitis in both tamarins infected with the recombinant GBV-B virus provides formal proof that GBV-B is a true hepatitis virus.

for HCV genotyping released in 1994, in addition to the highly conserved 5′-UTR and Core regions. Whereas treatment response/resistance was assessed mainly in the Interferon sensitivity determining region (ISDR) region of NS5A. Conclusions: While sequence analysis of HCV is generally constricted to certain regions of the HCV genome there is little consistency in the positioning of population sequencing primers, with the exception of a few highly referenced manuscripts. This study demonstrates the heterogeneity of HCV sequencing in the field, and provides a comprehensive database of previously published primer sets that may be utilised in future sequencing studies.

Hepatitis C virus (HCV) causes liver-related death in more than 300,000 people annually. Treatments for patients with chronic HCV are suboptimal, despite the introduction of directly acting antiviral agents. There is no vaccine that prevents HCV infection. Relevant animal models are important for HCV research and development of drugs and vaccines. Chimpanzees are the best model for studies of HCV infection and related innate and adaptive host immune responses. They can be used in immunogenicity and efficacy studies of HCV vaccines. The only small animal models of robust HCV infection are T- and B- cell deficient mice with human chimeric livers. Although these mice cannot be used in studies of adaptive immunity, they have provided new insights into HCV neutralization, interactions between virus and receptors, innate host responses, and therapeutic approaches. Recent progress in developing genetically humanized mice is exciting, but these models only permit studies of specific steps in the HCV life cycle and have limited or no viral replication.

Background & Aims Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-α. Methods Huh7.5 hepatoma cells were transfected with RNA of genotype 1–7 NS5A recombinants. Viability was determined by measuring HCV replication and infectivity titers. Putative adaptive mutations were analyzed by reverse genetics. The activity of antiviral agents was determined in high-throughput infection assays. Results Cells infected with viable HCV that expressed NS5A of genotypes 1–7 produced relatively high viral titers; most NS5A recombinants required introduction of specific adaptive mutations. The efficacy of the NS5A inhibitor BMS-790052 varied greatly, based on NS5A isolate, with median effective concentration (EC 50 ) values ranging from 0.009 nmol/L to 14 nmol/L; the high sensitivity of genotype 1b NS5A to BMS-790052 reflected observations from clinical studies. Specific residues in NS5A domain I were associated with >100-fold variations in sensitivity between isolates of the same HCV subtype. The Y/T2065H mutation conferred resistance to BMS-790052 that varied among NS5A isolates. When infected cultures were incubated with interferon-α, all NS5A recombinants had EC 50 values of ∼0.2 IU/mL, including an NS5A genotype 1b mutant with a putative sensitive-type, interferon sensitivity determining region. Conclusions We developed efficient in vitro systems in which recombinant viruses express HCV genotypes 1–7 NS5A; these permit genotype- and isolate-specific analyses of NS5A and the effects of antiviral compounds and resistance mutations. These culture systems will facilitate development of specific inhibitors against NS5A of different HCV variants.

J.M. Gottwein1,2, J.B. Lademann1,2, T.K.H. Scheel1,2, T.B. Jensen1,2, J.C. Prentoe1,2, J. Bukh1,2,3. 1Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital Hvidovre, Hvidovre, 2CO-HEP, Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 3Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA E-mail: judith@gottwein.eu